A kind of antibody-conjugated drug targeting EGFR, its preparation method and its application

A technology of antibody-drug coupling and antibody, which is applied in the direction of antibodies, drug combinations, anti-tumor drugs, etc., to achieve the effects of stable and controllable quality, reduced toxicity, and considerable yield

Active Publication Date: 2021-06-08
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, antibody therapy has certain limitations, and it is usually combined with chemotherapy drugs to achieve curative effect

Method used

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  • A kind of antibody-conjugated drug targeting EGFR, its preparation method and its application
  • A kind of antibody-conjugated drug targeting EGFR, its preparation method and its application
  • A kind of antibody-conjugated drug targeting EGFR, its preparation method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1V

[0052] The synthesis of embodiment 1VC-MMAE (MC-VC-PAB-MMAE):

[0053] 1.1 Synthesis of MMAE

[0054] Synthetic route such as figure 1 as shown in a:

[0055] Step a: Synthesis of Boc-Dap-N-desmethylephedrine

[0056] Compound Boc-Dap-OH (500mg, 1.7mmol) and (1S, 2R)-(±)-desmethylephedrine (289.3mg, 1.9mmol) were dissolved in 10mL DMF, followed by adding DEPC (368.9mg, 2.2 mmol ) and Et 3 N (228.8mg.2.2mmol), the reaction was stirred overnight at room temperature. After the reaction was completed, add water to stop the reaction, extract three times with ethyl acetate, and extract once with saturated NaCl solution, anhydrous NaCl 2 SO 4 Let dry overnight. Separation and purification by column chromatography, the separation condition was petroleum ether: ethyl acetate = 5:1, and 485 mg of white solid was obtained. C 23 h 36 N 2 o 5 .MS(ESI)m / z:321. 1 H NMR (400MHz, DMSO-d 6 )δ7.66(d,J=8.6Hz,1H),7.36–7.25(m,4H),7.22–7.16(m,1H), 5.37(d,J=4.6Hz,1H),4.46(t,J =5.1Hz,1H...

Embodiment 2

[0075] Example 2 Preparation of Antibody Drug Conjugate LR004-VC-MMAE

[0076] Include the following steps:

[0077] 1) The MC-VC-PAB-MMAE prepared in Example 1 (that is, the linker-cytotoxic drug) was dissolved in dimethyl sulfoxide to obtain a linker-cytotoxic drug stock solution.

[0078] 2) Dissolving TCEP reducing agent in buffer solution PBS (containing 1 mM DTPA), and preparing a certain molar amount of reducing agent stock solution.

[0079] 3) Replace the buffer of LR004 antibody before reduction, and the buffer system is PBS (containing 1mM DTPA). The replacement method is to replace the desalting column. The replacement method is as follows: load 2.5 mL of sample on the desalting column, elute 3.5 mL with replacement buffer, concentrate the concentration by ultrafiltration at 4000 rpm, and centrifuge at 4° C. for 10 min. Configure to obtain 5mg / mL LR004 antibody solution.

[0080] 4) Mix the 5 mg / mL LR004 antibody solution in step 3) with the reducing agent TCEP...

Embodiment 3

[0086] Example 3 Detection of EGFR expression in different tumor cells

[0087] 3.1 Western Blot detection of EGFR expression levels in different tumor cells

[0088] Collect logarithmic phase esophageal cancer cells (KYSE450), breast cancer cells (MDA-MB-468), head and neck cancer cells (SCC-25), colon cancer cells (HCT-116), pancreatic cancer cells (AsPC-1), Osteosarcoma cells (143B) and prostate cancer cells (PC-3). Cells were digested to collect cells and counted, collecting 1 × 10 7 The above cells were centrifuged and the supernatant was discarded for later use. Pre-cool the RIPA protein extraction reagent, add protease inhibitors (phosphorylated proteins need to add phosphatase inhibitors at the same time). Add 0.1M PMSF stock solution before the start of protein extraction, and the final concentration of PMSF is 1mM. For cell counting, the number of cells is 1 x 10 7 Add 1ml of lysate, centrifuge at 4°C, store, and measure protein concentration by BCA method. The...

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Abstract

The invention discloses an antibody-coupled drug targeting EGFR, a preparation method and an application thereof. The antibody-drug conjugate targeting EGFR is named LR004-VC-MMAE, which consists of an antibody, a cytotoxic drug and a linker. The antibody-drug conjugate has a structure shown in Formula I, wherein, mAb is LR004 monoclonal antibody, n=2-8. The novel antibody-conjugated drug LR004‑VC‑MMAE can not only target the EGFR antigen, but also has a strong activity of killing tumor cells. And compared with LR004 itself, it does not affect the affinity, endocytic activity and targeting of the antibody, and better retains its biological function. Compared with LR004, the anti-tumor effect of LR004-VC-MMAE antibody conjugate drug was significantly improved, and the tumor disappeared. Compared with LR004, LR004‑VC‑MMAE antibody-drug conjugated total antibody has a longer half-life, slower clearance rate, lower concentration of free MMAE in plasma, short half-life, and faster clearance rate, which is beneficial to reduce toxicity.

Description

technical field [0001] The invention belongs to the field of biotechnology medicine. Specifically, the present invention provides a class of antibody-coupled drugs that can produce targeted tumor killing effects, their preparation methods and uses. [0002] technical background [0003] According to the "Global Cancer Report", in 2018, it is estimated that there will be 18.1 million new cancer cases and 9.6 million deaths in the world, with an average of 18 cancer deaths every minute, and the global cancer burden will further increase. China is a big cancer country, and the number of new cases is on the rise. Among them, solid tumors such as lung cancer, breast cancer, and colorectal cancer are cancers with a very high incidence rate, and usually when diagnosed, the disease has already developed to an advanced stage. At present, the main treatment methods are surgery, radiation therapy and drug chemotherapy. This treatment strategy not only has a poor prognosis and a low 5-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/68A61K47/65A61K47/55A61K38/07A61K39/395A61P35/00
CPCA61K38/07A61K39/39558A61K47/55A61K47/65A61K47/6813A61K47/6849A61P35/00
Inventor 李卓荣胡馨月苗庆芳王蓉金洁刘秀均崔阿龙李毅甄永苏
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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